Physiological, molecular, and genetic mechanism of action of the biostimulant Quantis™ for increased thermotolerance of potato (Solanum tuberosum L.)

Background: Raising global temperatures limit crop productivity and new strategies are needed to improve the resilience of thermosensitive crops such as potato (Solanum tuberosum L.). Biostimulants are emerging as potential crop protection products against environmental stress, however their mechanism of action remains largely unknown, hindering their wider adoption. We used comprehensive physiological, molecular, and mass spectrometry approaches to develop understanding of the mechanism of plant thermotolerance exerted by the biostimulant, Quantis™, under heat stress. Using orthologues gene mutations in Arabidopsis thaliana we report heat-defence genes, modified by Quantis™, which were also investigated for potential overlapping functions in biotic stress defence to Sclerotinia sclerotiorum and Rhizoctonia solani. Results: Quantis™ enhanced PSII photochemical efficiency and decreased thermal dissipation of potato grown under heat stress. These effects were associated with upregulation of genes with antioxidant function, including PR10, flavonoid 3′‐hydroxylase and β-glucosidases, and modulation of abscisic acid (ABA) and cytokinin (CK) activity in leaves by Quantis™. The biostimulant modulated the expression of the heat-defence genes, PEN1, PR4 or MEE59, with functions in leaf photoprotection and root thermal protection, but with no overlapping function in biotic stress defence. Protective root growth under heat stress, following the biostimulant application, was correlated with enhanced CK signalling in roots. Increased endogenous concentrations of ABA and CK in potato leaves and significant upregulation of StFKF1 were consistent with tuberisation promoting effects. Quantis™ application resulted in 4% tuber weight increase and 40% larger tuber size thus mitigating negative effects of heat stress on tuber growth. Conclusions: Quantis™ application prior to heat stress effectively primed heat tolerance responses and alleviated temperature stress of S. tuberosum L. and A. thaliana by modulating the expression and function of PR4 and MEE59 and by regulating CK activity above and below ground, indicating that the mechanism of action of the biostimulant is conserved, and will be effective in many plant species. Thus, a biostimulant application targeting the most susceptible crop developmental stages to heat disorders can be effectively integrated within future agronomy practices to mitigate losses in other thermosensitive crops

An oxygen-sensing mechanism for angiosperm adaptation to altitude

Flowering plants (angiosperms) can grow at extreme altitudes, and have been observed growing as high as 6,400 metres above sea level1,2; however, the molecular mechanisms that enable plant adaptation specifically to altitude are unknown. One distinguishing feature of increasing altitude is a reduction in the partial pressure of oxygen (pO2). Here we investigated the relationship between altitude and oxygen sensing in relation to chlorophyll biosynthesis—which requires molecular oxygen3—and hypoxia-related gene expression. We show that in etiolated seedlings of angiosperm species, steady-state levels of the phototoxic chlorophyll precursor protochlorophyllide are influenced by sensing of atmospheric oxygen concentration. In Arabidopsis thaliana, this is mediated by the PLANT CYSTEINE OXIDASE (PCO) N-degron pathway substrates GROUP VII ETHYLENE RESPONSE FACTOR transcription factors (ERFVIIs). ERFVIIs positively regulate expression of FLUORESCENT IN BLUE LIGHT (FLU), which represses the first committed step of chlorophyll biosynthesis, forming an inactivation complex with tetrapyrrole synthesis enzymes that are negatively regulated by ERFVIIs, thereby suppressing protochlorophyllide. In natural populations representing diverse angiosperm clades, we find oxygen-dependent altitudinal clines for steady-state levels of protochlorophyllide, expression of inactivation complex components and hypoxia-related genes. Finally, A. thaliana accessions from contrasting altitudes display altitude-dependent ERFVII activity and accumulation. We thus identify a mechanism for genetic adaptation to absolute altitude through alteration of the sensitivity of the oxygen-sensing system

A Yeast-Based Functional Assay to Study Plant N-Degron – N-Recognin Interactions

The N-degron pathway is a branch of the ubiquitin-proteasome system where amino-terminal residues serve as degradation signals. In a synthetic biology approach, we expressed ubiquitin ligase PRT6 and ubiquitin conjugating enzyme 2 (AtUBC2) from Arabidopsis thaliana in a Saccharomyces cerevisiae strain with mutation in its endogenous N-degron pathway. The two enzymes re-constitute part of the plant N-degron pathway and were probed by monitoring the stability of co-expressed GFP-linked plant proteins starting with Arginine N-degrons. The novel assay allows for straightforward analysis, whereas in vitro interaction assays often do not allow detection of the weak binding of N-degron recognizing ubiquitin ligases to their substrates, and in planta testing is usually complex and time-consuming

ERFVII action and modulation through oxygen-sensing in Arabidopsis thaliana

Oxygen is a key signalling component of plant biology, and whilst an oxygen-sensing mechanism was previously described in Arabidopsis thaliana, key features of the associated PLANT CYSTEINE OXIDASE (PCO) N-degron pathway and Group VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factor substrates remain untested or unknown. We demonstrate that ERFVIIs show non-autonomous activation of root hypoxia tolerance and are essential for root development and survival under oxygen limiting conditions in soil. We determine the combined effects of ERFVIIs in controlling gene expression and define genetic and environmental components required for proteasome-dependent oxygen-regulated stability of ERFVIIs through the PCO N-degron pathway. Using a plant extract, unexpected amino-terminal cysteine sulphonic acid oxidation level of ERFVIIs was observed, suggesting a requirement for additional enzymatic activity within the pathway. Our results provide a holistic understanding of the properties, functions and readouts of this oxygen-sensing mechanism defined through its role in modulating ERFVII stability

Mitochondrial retrograde signaling through UCP1-mediated inhibition of the plant oxygen-sensing pathway

Mitochondrial retrograde signaling is an important component of intracellular stress signaling in eukaryotes. UNCOUPLING PROTEIN (UCP)1 is an abundant plant inner-mitochondrial membrane protein with multiple functions including uncoupled respiration and amino-acid transport1,2 that influences broad abiotic stress responses. Although the mechanism(s) through which this retrograde function acts is unknown, overexpression of UCP1 activates expression of hypoxia (low oxygen)-associated nuclear genes.3,4 Here we show in Arabidopsis thaliana that UCP1 influences nuclear gene expression and physiological response by inhibiting the cytoplasmic PLANT CYSTEINE OXIDASE (PCO) branch of the PROTEOLYSIS (PRT)6 N-degron pathway, a major mechanism of oxygen and nitric oxide (NO) sensing.5 Overexpression of UCP1 (UCP1ox) resulted in the stabilization of an artificial PCO N-degron pathway substrate, and stability of this reporter protein was influenced by pharmacological interventions that control UCP1 activity. Hypoxia and salt-tolerant phenotypes observed in UCP1ox lines resembled those observed for the PRT6 N-recognin E3 ligase mutant prt6-1. Genetic analysis showed that UCP1 regulation of hypoxia responses required the activity of PCO N-degron pathway ETHYLENE RESPONSE FACTOR (ERF)VII substrates. Transcript expression analysis indicated that UCP1 regulation of hypoxia-related gene expression is a normal component of seedling development. Our results show that mitochondrial retrograde signaling represses the PCO N-degron pathway, enhancing substrate function, thus facilitating downstream stress responses. This work reveals a novel mechanism through which mitochondrial retrograde signaling influences nuclear response to hypoxia by inhibition of an ancient cytoplasmic pathway of eukaryotic oxygen sensing

The Cys-Arg/N-end rule pathway is a general sensor of abiotic stress in flowering plants

Abiotic stresses impact negatively on plant growth, profoundly affecting yield and quality of crops. Although much is known about plant responses, very little is understood at the molecular level about the initial sensing of environmental stress. In plants, hypoxia (low oxygen, which occurs during flooding) is directly sensed by the Cys-Arg/N-end rule pathway of ubiquitin-mediated proteolysis, through oxygen-dependent degradation of group VII Ethylene Response Factor transcription factors (ERFVIIs) via amino-terminal (Nt-) cysteine [1, 2]. Using Arabidopsis (Arabidopsis thaliana) and barley (Hordeum vulgare), we show that the pathway regulates plant responses to multiple abiotic stresses. In Arabidopsis, genetic analyses revealed that response to these stresses is controlled by N-end rule regulation of ERFVII function. Oxygen sensing via the Cys-Arg/N-end rule in higher eukaryotes is linked through a single mechanism to nitric oxide (NO) sensing [3, 4]. In plants, the major mechanism of NO synthesis is via NITRATE REDUCTASE (NR), an enzyme of nitrogen assimilation [5]. Here, we identify a negative relationship between NR activity and NO levels and stabilization of an artificial Nt-Cys substrate and ERFVII function in response to environmental changes. Furthermore, we show that ERFVIIs enhance abiotic stress responses via physical and genetic interactions with the chromatin-remodeling ATPase BRAHMA. We propose that plants sense multiple abiotic stresses through the Cys-Arg/N-end rule pathway either directly (via oxygen sensing) or indirectly (via NO sensing downstream of NR activity). This single mechanism can therefore integrate environment and response to enhance plant survival

A phloem‐localized Arabidopsis metacaspase (AtMC3) improves drought tolerance

Increasing drought phenomena pose a serious threat to agricultural productivity. Although plants have multiple ways to respond to the complexity of drought stress, the underlying mechanisms of stress sensing and signaling remain unclear. The role of the vasculature, in particular the phloem, in facilitating inter-organ communication is critical and poorly understood.Combining genetic, proteomic and physiological approaches, we investigated the role of AtMC3, a phloem-specific member of the metacaspase family, in osmotic stress responses in Arabidopsis thaliana. Analyses of the proteome in plants with altered AtMC3 levels revealed differential abundance of proteins related to osmotic stress pointing into a role of the protein in water-stress-related responses.Overexpression of AtMC3 conferred drought tolerance by enhancing the differentiation of specific vascular tissues and maintaining higher levels of vascular-mediated transportation, while plants lacking the protein showed an impaired response to drought and inability to respond effectively to the hormone abscisic acid.Overall, our data highlight the importance of AtMC3 and vascular plasticity in fine-tuning early drought responses at the whole plant level without affecting growth or yield.ISSN:0028-646XISSN:1469-813

Oxygen-dependent proteolysis regulates the stability of angiosperm polycomb repressive complex 2 subunit VERNALIZATION 2

The polycomb repressive complex 2 (PRC2) regulates epigenetic gene repression in eukaryotes. Mechanisms controlling its developmental specificity and signal-responsiveness are poorly understood. Here, we identify an oxygen-sensitive N-terminal (N-) degron in the plant PRC2 subunit VERNALIZATION(VRN) 2, a homolog of animal Su(z)12, that promotes its degradation via the N-end rule pathway. We provide evidence that this N-degron arose early during angiosperm evolution via gene duplication and N-terminal truncation, facilitating expansion of PRC2 function in flowering plants. We show that proteolysis via the N-end rule pathway prevents ectopic VRN2 accumulation, and that hypoxia and long-term cold exposure lead to increased VRN2 abundance, which we propose may be due to inhibition of VRN2 turnover via its N-degron. Furthermore, we identify an overlap in the transcriptional responses to hypoxia and prolonged cold, and show that VRN2 promotes tolerance to hypoxia. Our work reveals a mechanism for post-translational regulation of VRN2 stability that could potentially link environmental inputs to the epigenetic control of plant development